Rabbit anti afp

Livers and liver tumors were dissected and snap frozen in liquid nitrogen. Snap frozen tissues were homogenized in Cell Lysis Buffer (Cell Signaling Technology) supplemented with complete protease inhibitors (Roche). Expression of AFP was analyzed by western blot analysis using standard procedures. Rabbit anti-AFP (1/1000, Abcam) and rabbit anti alpha-actin (1/1000) diluted in PBS with 0.5% (w/v) nonfat dry milk and 0.2% (v/v) Triton X100 were used as primary antibodies. Detection of proteins was carried out with the ECL method using the Western Lightning enhanced luminol-based chemiluminescence HRP substrate (Perkin Elmer).

Samples were washed with PBS, fixed with 4% paraformaldehyde solution (Wako Pure Chemical Industries) for 20 min, permeabilized with 1% Triton™ X-100 (Sigma-Aldrich) in PBS for 20 min, and then stained overnight with the following antibodies: phycoerythrin (PE) mouse anti-human NANOG, Alexa Fluor^® 488 mouse anti-OCT3/4 (BD Biosciences), mouse anti-TUJ1 (β-tubulin III; Sigma-Aldrich), rabbit anti-α-SMA (PA1-37024, Thermo Fisher Scientific), and rabbit anti-AFP (Abcam, Cambridge, UK). The secondary antibodies Alexa Fluor^® 488 goat anti-mouse IgG (Thermo Fisher Scientific) and Alexa Fluor^® 568 Goat Anti-Rabbit IgG (Thermo Fisher Scientific) were then added. The nuclei were stained with Hoechst 33342 (Lonza).

Mouse livers were fixed in neutral buffered formalin for 24 h at room temperature and then embedded and processed according to standard protocols. Liver sections were deparaffinized through graded ethanol solutions. After an antigen retrieval procedure of 30 min, sections were stained with specific antibodies using the avidin-biotin complex system (Vector Laboratories, SP-2001, PK-6100). 3,3’-diaminobenzidine (DAB, Vector Laboratories, SP-4105) was used as the substrate. Cell nuclei were counterstained with hematoxylin. Primary antibodies against AFP (Rabbit anti-AFP, Abcam, Cat# ab46799, dilution 1:500 v/v) and Ki67 (mouse anti-Ki67 B56, BD, Cat# 556003, dilution 1:500 v/v) were involved in this section.